Gastrointestinal cancer is a significant health problem, ranking fourth in incidence and second in death in the United States. Abnormal differentiation and increased proliferation are hallmarks of carcinogenesis. The molecular mechanisms that regulate cellular proliferation and differentiation in gastrointestinal development are far from being completely understood. We have shown by gene targeting that the transcription factor Foxl1 (previously Fkh6) is essential in the control of proliferation in the gastrointestinal epithelium. As Foxl1 is restricted to the mesenchyme of the gastrointestinal tract, but the phenotype is expressed in the epithelium, we postulate that Foxl1 regulates a signaling pathway between the two cell layers. We have shown by biochemical means that the Wnt/APC/Beta-catenin pathway is activated in Foxl1 mutant mice. We hypothesize that mutations in Foxl1 synergize genetically with those in the WntIAPC/Beta-catenin pathway to promote tumor initiation or progression in the GI tract.This hypothesis will be addressed by interrelated Specific Aims that employ advanced mouse genetics and mutational analysis in man to explore the role of Foxl1 and its targets in gastrointestinal proliferation and differentiation. First, we will test our hypothesis that Foxl1 is a genetic modifier of the Wnt/APC/Beta-catenin pathway by histological and biochemical analyses of mice carrying mutations in both Foxl1 and Min (the mouse APC gene). In addition, we will perform mutation analysis for FOXL1 in patients with APC mutations to investigate whether FOXL1 can act as a modifier in APC-dependent carcinogenesis. Second, we will investigate the role of the Foxl1 target Syndecan-1 in epithelial proliferation in the gut through gain-of-function and loss-of-function experiments. Third, we will analyze the role of the Zinc-finger protein Klf4 (Kruppel-like factor 4) in gastrointestinal development by loss-of-function and gain-of-function experiments. Klf4 is expressed in the gastrointestinal epithelium and has been shown previously to be a Foxl1 target. Due to the role of Klf4 in skin barrier function we will employ tissue-specific gene ablation to study KLf4 function in the adult gastrointestinal tract. Together, these studies will further our understanding of the regulatory circuits that control gastrointestinal proliferation in normal development and carcinogenesis.